Modern Pathology (2010) 23, S52–S59 S52 & 2010 USCAP, Inc. All rights reserved 0893-3952/10 $32.00

Issues and updates: evaluating estrogen receptor-a, progesterone receptor, and HER2 in breast cancer

D Craig Allred

Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO, USA

There are currently three prognostic/predictive biomarkers used in routine clinical management of patients with breast cancer, and their assessment is mandatory. They include estrogen receptor-alpha (ERa), progesterone receptor (PgR), and the HER2 oncogene/oncoprotein. This paper briefly reviews the assessment of ERa, PgR, and HER2 in breast cancer, emphasizing recent progress and persistent controversies. Modern Pathology (2010) 23, S52–S59; doi:10.1038/modpathol.2010.55

Keywords: breast cancer; immunohistochemistry; estrogen receptor; progesterone receptor; HER2

Immunohistochemistry (IHC) has an important role and new methodologies). HER2 is further along than in the assessment of prognostic and predictive hormone receptors on many of these issues, and will factors in invasive breast cancer (IBC) today. Prog- be discussed first. nostic factors are defined as clinical, pathological, and biological features associated with the innate aggressiveness of untreated IBCs and, if adverse HER2 oncogene/oncoprotein enough, usually result in the use of additional (ie, adjuvant) therapies following surgery. Predictive HER2 (also referred to as HER2/neu and erbB2)is factors, in contrast, are defined as features that a proto-oncogene located on chromosome 17.9 It predict the likelihood of responding to specific encodes a tyrosine-kinase receptor residing on the types of adjuvant therapies. Many features have surface membrane of breast epithelial cells.10 HER2 both prognostic and predictive significance to forms complexes with similar proteins (such as varying degrees. Although a large number of erbB1, erbB3, and erbB4), which act as receptors for potentially useful factors have been identified,1–4 several ligands (such as epidermal growth factor, only three are currently used in routine clinical heregulin, and amphiregulin), which regulate many practice and their assessment is mandatory. These normal cellular functions, including proliferation, include the estrogen receptor-alpha (ERa), the survival, and apoptosis.11–13 Many studies during progesterone receptor (PgR), and the HER2 onco- the past 25 years have shown that the HER2 gene is gene/oncoprotein. IHC is the most commonly used amplified in up to 30% IBCs, and that amplification method of assessing these factors, although fluor- is highly correlated with overexpression of the escent in situ hybridization (FISH) also has a protein.11,12 The rate is closer to 15% today, which prominent role in HER2 testing.5–8 This presentation is probably because of screening mammography briefly reviews the assessment of these biomarkers detecting early-stage tumors before amplification in breast cancer, with special emphasis on standar- has occurred. dization, validation, and other issues of importance The relationship between HER2 status and clin- during the past 5 years (such as new clinical ical outcome is complex, and varies with the setting. applications, testing error rates, testing guidelines, There is a weak but significant association between poor outcome and ‘positive’ (ie, amplified and/or overexpressed) HER2 in patients receiving no addi- Correspondence: Professor DC Allred, MD, Department of tional therapy after initial surgery, which represent a Pathology and Immunology, Washington University School of small minority of patients today.14,15 Most patients Medicine, 660 S. Euclid Avenue, Campus Box 8118, St Louis, MO 63110, USA. receive some type of adjuvant therapy, and the E-mail: [email protected] association between HER2 status and outcome Received 25 January 2010; accepted 26 January 2010 seems to depend on the type of therapy.14–22 For

www.modernpathology.org Evaluating ERa, PgR, and HER2 in breast cancer DC Allred S53 example, many studies suggest that HER2-positive implemented only 2 years ago, studies are beginning IBCs are resistant to certain types of cytotoxic to show that they have resulted in substantial chemotherapies (eg, the combination of cytoxan- improvement of testing accuracy.34 Figure 1 high- methotrexate-5-fluoracil) but sensitive to others (eg, lights the history, assays, clinical utility, problems, anthracyclines and taxanes). Other studies suggest and solutions represented by the ASCO/CAP testing that positive HER2 status may be associated with guidelines for HER2 testing. resistance to hormonal therapies, although not all agree and this issue remains somewhat controver- sial.21,23 The most promising and useful findings Estrogen receptor-a come from recent studies showing that HER2- positive tumors respond favorably to new anti- ERa is as a nuclear transcription factor activated by body-based therapies, which specifically target the estrogen to regulate growth and differentiation of HER-2 protein, such as trastuzumab,22,24 and the normal breast epithelial cells.35–37 These pathways main reason for assessing HER-2 status today is to remain operative to varying degrees in IBCs, includ- identify candidates for targeted therapy. Although ing estrogen-stimulated growth of tumor cells trastuzumab was originally demonstrated as being expressing ERa, which is detrimental.36–38 ERa effective in HER2-positive metastatic disease, more expression has been measured in IBCs for almost recent clinical trials have demonstrated significant 40 years. During the first 20–25 years, it was benefit as adjuvant therapy for women with less measured by radiolabeled biochemical ligand (ie, advanced HER2-positive breast cancer.25–28 For ex- estrogen)-binding assays (LBAs) on whole tissue ample, the NSABP-B31 clinical trial, which rando- extracts prepared from fresh-frozen tumor samples, mized patients with HER2-positive breast cancer to which was costly and difficult. Many studies using adjuvant chemotherapy±trastuzumab, showed a LBAs in large randomized clinical trials showed that 52% improvement in disease-free survival with ERa was a relatively weak prognostic factor but a trastuzumab, which is remarkable. very strong predictive factor for response to hormo- There has been a long and persistent controversy nal therapies, such as tamoxifen.38 Tamoxifen, about whether it is best to evaluate HER2 status by which binds ERa and blocks estrogen-stimulated measuring protein expression by IHC or gene growth, has been shown to significantly reduce amplification by FISH. Although there are vocal disease recurrence and prolong life in patients with proponents of both methods, many studies have ERa-positive IBCs.38,39 The clinical response to shown that, when properly performed, there is a newer types of hormonal therapies, such as the very strong correlation between IHC and aromatase inhibitors, which suppress the produc- FISH,8,15,29,30 and that they are equivalent (and tion of estrogen, is also dependent on the status of sometimes complimentary) in clinical utility. ERa, and only positive tumors benefit.40–42 The Approximately 70% of breast cancers show little primary reason for assessing ERa is its ability to or no protein expression, a normal gene copy predict response to these hormonal therapies. number, and do not respond to trastuzumab. Although the clinical utility of assessing ERa was Another roughly 15% show low-to-intermediate initially based almost entirely on studies using levels of protein expression, the gene is amplified technically standardized LBAs, beginning in the (usually at low levels) in about a third of these cases, early 1990s, laboratories around the world aban- and there is still uncertainty regarding how well this doned LBAs in favor of IHC, which is used for group responds. The remaining 15% of cases show nearly all testing today. very strong membrane staining, indicating high There are advantages to using IHC over LBAs, levels of protein expression, the gene is almost especially its ability to measure ERa on routine always amplified in these tumors, and they show the formalin-fixed paraffin-embedded samples, elimi- best response in any setting to trastuzumab, as well nating the need for fresh-frozen samples and the as newer and more effective therapies targeting onerous infrastructure required to provide it. Other HER2.16,31 advantages include lower cost, better safety, as well A particularly notable recent issue regarding as superior sensitivity and specificity in the sense HER2 testing is the joint publication of guidelines that the assessment of ERa is restricted to tumor for HER2 testing by the American Society of Clinical cells under direct microscopic visualization, inde- Oncologists (ASCO) and College of American pendent of tumor cellularity or the presence of Pathologists (CAP).8 They were developed to im- benign epithelium, which is problematic for LBAs. prove substantial inaccuracies in HER2, which were For all these reasons and more, IHC was approved revealed primarily in association with large clinical by the CAP and ASCO for routine clinical use.5,6 trials in which results from laboratories of enrolling However, despite these approvals, there are signifi- institutions were compared with testing by expert cant problems with IHC that persist today, including central laboratories. They consisted of false-negative the widespread use of diverse staining procedures of and false-positive IHC results up to 20%,32,33 and unequal quality and varied often arbitrary methods false-positive FISH results up to 15%,33 which are of interpreting results, resulting in error rates as high all unacceptable. Although the guidelines were as 20% overall (primarily false negatives).43–49 There

Modern Pathology (2010) 23, S52–S59 Evaluating ERa, PgR, and HER2 in breast cancer S54 DC Allred

ASCO/CAP Guidelines HER2 Protein for HER2 Testing in Breast Cancer Arch Pathol Lab Med 131:18 and J Clin Oncol 25:118, 2007

Pre-clinical Studies Invasive Breast Cancer (Mandatory)

Test with Validated Assay 1987 – 1st Clinical Study Science, 1987 IHC (>30% cells) or FISH (#genes/chromosome)

Many Assays Protein IHC Positive Equivocal Negative RNA FISH intensity 3+ intensity 2+ intensity 0/1+ DNA ratio >2.2 ratio 1.8 to 2.2 ratio < 1.8 ≈ ↑ Amp Exp (∼15% cases) (∼15% cases) (∼70% cases)

Many Purposes

25 Years Prognostic Untreated Retest with Validated Assay Predictive Chemotherapy IHC to FISH Hormonal Therapy FISH to IHC Targeted Therapy Trastuzumab Newer Drugs Positive Equivocal Negative ratio > 2.2 ratio 1.8 to 2.2 ratio < 1.8 Problems ∼20% Errors intensity 3+ intensity 2+ intensity 0/1+ ∼ ∼ Today Solutions ( 5% cases) (? % cases) ( 10% cases) ASCO/CAP Guidelines

Figure 1 Overview of the history, assays, and clinical utility of HER2 in breast cancer, as well as problems with testing accuracy and solutions provided by the ASCO/CAP testing guidelines for HER2 testing.

are currently no widely accepted solutions to these significant benefit far above that of ERa-negative problems, but most can be avoided by following tumors, which are essentially unresponsive. This general guidelines which have been published for evidence provides support for laboratories adopting assessing prognostic and predictive factors6,50,51 Z1% positive staining for tumor cells as the These guidelines all agree that tests used in routine definition of ‘ERa-positive’ clinically and setting clinical practice should be based on sensitive and the threshold higher may deny hormonal therapy to specific reagents, standardized laboratory proce- some patients who might benefit, and a 1% cutoff dures and, especially, calibrated to relevant clinical has now been clinically validated in several com- outcome in a comprehensive manner. prehensive studies.41,52–58 In head-to-head compar- There are arguably no tests for any prognostic or isons, many studies have also shown that assessing predictive biomarkers in breast or any other types of ERa by IHC provides equivalent or even stronger cancers, which entirely satisfy these guidelines, but correlations with response to hormonal therapy than several strategies have been published for assessing LBAs,53,60 which is comforting as IHC replaced LBA ERa by IHC which come very close.41,52–58 Collec- before such proof was available. tively, these studies show that B75% of IBCs A few recent studies have suggested that the express ERa, that it is almost entirely nuclear in distribution of ERa assessed by IHC in IBC is location, and that there is tremendous variation essentially bimodal (either entirely negative or between ERa-expressing tumors on a continuum strongly positive), leading the authors to recom- ranging from 0 to nearly 100% positive cells.59 More mend reporting results as either positive or negative importantly, they show a direct correlation between without further quantification.61,62 However, the the likelihood of clinical response to hormonal studies reporting bimodal ERa are not an accurate therapies and the level of ERa expression.53 representation of the true biological continuum of Although there is a gradient of increasing response expression, and may be too sensitive, resulting in with increasing levels of ERa, the gradient is skewed saturated signals.63 It is important to provide such that tumors expressing even very low levels quantitative ERa results for many reasons. Foremost (eg, between 1 and 10% positive cells) show a among them, most patients want to know their

Modern Pathology (2010) 23, S52–S59 Evaluating ERa, PgR, and HER2 in breast cancer DC Allred S55 predicted outcome as precisely as possible and their measured by standardized LBAs for nearly two physicians use quantitative information in making decades and shown to be a weak prognostic factor therapeutic decisions. For example, recent results but a relatively strong predictive factor for response from clinical trials suggest that most postmenopau- to hormonal therapy. LBAs for PgR were replaced sal patients with tumors expressing very high levels by IHC beginning in the mid-1990s, and IHC of ERa can be optimally treated with adjuvant was eventually approved by the CAP and ASCO hormonal therapy alone, and can safely forego the for routine clinical use despite persistent short- rigors of chemotherapy, which is an important comings.5–7 recent improvement in medical care.64,65 Compared with ERa, there are fewer studies in the Assessing ERa by IHC may also be useful in medical literature standardizing and validating IHC patients with ductal carcinoma in situ (DCIS). assays for PgR.54,56,57,60,72 Those available show that Results from a large randomized clinical trial PgR is expressed in the nuclei of 60–70% of IBCs, (NSABP-B24) showed that, in patients with DCIS that expression varies on a continuum ranging managed by lumpectomy and postoperative radia- from 0 to nearly 100% positive cells, that there is a tion, the use of tamoxifen resulted in an additional direct correlation between PgR levels and res- 50% relative reduction in local recurrence in ERa- ponse to hormonal therapies, and that tumors with positive disease, and assessing ERa by IHC in DCIS even very low levels of PgR-positive cells (Z1%) is now routine in many centers.66,67 have a significant chance of responding.54,72 Pre- The most notable current issue related to ERa liminary studies suggest that, similar to ERa, PgR testing by IHC is the soon-to-be published guide- expression is also associated with reduced local lines by ASCO/CAP to improve accuracy.58 These recurrence in patients with DCIS treated with guidelines are conceptually modeled after the lumpectomy and radiation followed by hormonal recently published guidelines for HER2 testing by therapy.66,67 ASCO/CAP,8 which have already shown a positive Although the expression of PgR is highly corre- impact on quality.34 Hopefully, the new guidelines lated with ERa, the correlation is imperfect, result- for ERa (and PgR) testing by IHC will be as helpful, ing in four possible phenotypes of combined and following them will be mandatory for labora- expression, each with significantly different rates tories conducting the tests under CAP certification. of response to hormonal therapy, which would not Figure 2 outlines essential general elements of be apparent measuring one or the other alone. For accurate testing for ERa (and PgR) in breast cancer example, in a recent comparison of patients receiv- by IHC. ing adjuvant tamoxifen, the relative risk of disease Another outcome partially motivated by proble- recurrence was 28% higher in patients with ERa- matic IHC testing is the development and ongoing positive/PR-negative than ERa-positive/PgR-posi- validation of newer technologies to assess ERa and tive tumors.73,74 Distinguishing these significantly other clinically relevant gene products simulta- different outcomes is the primary reason that both neously, including qRT-PCR (eg, OncotypeDX)46,68 ERa and PgR are measured in routine clinical and gene-expression microarrays.69 Eventually, practice. these multigene prognostic and predictive signa- Recent studies75–80 have suggested that functional tures will replace ERa testing by IHC, because the ERa, which is predominately nuclear in location in response to hormonal therapies is biologically too most IBCs, may also reside at the outer cell complex to be accurately predicted by measuring a membrane in a subset of tumors, especially those single gene, regardless of how it is performed. that are HER2 positive. The majority of HER-positive However, these new tests are still being validated IBCs are also PgR negative, suggesting that nuclear and are not mature enough to be used in routine ERa may be nonfunctional in these cases and, thus, clinical practice; therefore, testing for ERa by IHC possibly unresponsive to the antagonistic effects of will be with us for a while longer (perhaps a tamoxifen. However, membrane ERa appears to decade). remain functional and promotes tumor cell prolif- eration in cooperation with overexpressed HER2. To further complicate the story, there is also evidence Progesterone receptor that tamoxifen has a stimulatory or agonist affect on membrane ERa, leading to the speculation that PgR is also routinely assessed by IHC in IBCs. ERa aromatase inhibitors may remain effective in this regulates the expression of PgR; hence, the presence setting as they inhibit the upstream production of of PgR usually indicates that the estrogen-ERa estrogen, which is the ligand for both nuclear and pathway is intact and functional.35,38,70,71 Once membrane ERa. If these preliminary studies are expressed, PgR is activated by the hormone proges- confirmed, then the quantitative assessment of PgR terone to help regulate several important normal may take on added importance, especially in the cellular functions, including proliferation which, of ERa/erbB2-positive subset of IBCs. course, is detrimental in breast cancers.35,38,70,71 As with ERa, the most notable current issue for Most of the discussion above regarding the historical assessing PgR by IHC is the increasing alarm about assessment of ERa in IBCs also applies to PgR. It was problems with accuracy and the impending ASCO/

Modern Pathology (2010) 23, S52–S59 Evaluating ERa, PgR, and HER2 in breast cancer S56 DC Allred

Essential Elements of Accurate Testing for ERα and PgR by IHC Note: Defer to ASCO/CAP Guidelines (In Press)

IBCs (Mandatory) and DCIS (Optional)

Validated IHC Assays Negative < 1% cells Positive Quantify results ≥ 1% cells No Endocrine Therapy Quantify results ∼ Expect 25% ER and 35% PgR Endocrine Therapy Confirm/retest if: Expect ∼75% ER and 65% PgR Neg. internal/external controls Low histological grade Lobular subtype Tubular subtype Mucinous subtype ∼ Other… ∼1-2% 10%

< 1% ∼30% ∼60% ∼100%

Figure 2 Overview of essential elements required for accurate testing of ERa and PgR status in breast cancer by immunohistochemistry. Similar to HER2 testing, guidelines have recently been developed by the ASCO/CAP to reduce the error rate associated with testing (in press; Arch Pathol Lab Med and J Clin Oncol), estimated to be as high as 20% overall.

CAP guidelines intended to improve it. Alternative cancer. All are targets and/or indicators of response methods for assessing PgR are also emerging, to highly effective therapies in many clinical including qRT-PCR (eg, Oncotype DX). However, settings, so accurate assessment is essential. How- measuring PgR by IHC will also be with us for several ever, accurate testing has been problematic (with years; therefore, improving accuracy is essential. error rates of Z20% with all of them), and there are several recent and ongoing efforts to improve it, such as the recently published ASCO/CAP guide- Summary lines for HER2 testing, and imminent similar guide- lines for ERa and PgR.58 It is the responsibility of The assessment of ERa, PgR, and HER2 are manda- every pathologist evaluating these biomarkers to be tory in the routine care of all patients with breast aware of these issues, to possess appropriate

Modern Pathology (2010) 23, S52–S59 Evaluating ERa, PgR, and HER2 in breast cancer DC Allred S57

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